Browsing by Subject "TOXIN"

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  • Worbs, Sylvia; Skiba, Martin; Söderström, Martin; Rapinoja, Marja-Leena; Zeleny, Reinhard; Russmann, Heiko; Schimmel, Heinz; Vanninen, Paula; Fredriksson, Sten-Åke; Dorner, Brigitte G. (2015)
    Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon gold standards are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization-time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.
  • Nyholm, Outi; Halkilahti, Jani; Wiklund, Gudrun; Okeke, Uche; Paulin, Lars; Auvinen, Petri; Haukka, Kaisa; Siitonen, Anja (2015)
    Background Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor. Methods The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied. Results The human STEC/ETEC strains clustered with strains representing ETEC, STEC, entero-aggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only. Conclusions This study shows that pathogroup-associated virulence genes of different E. coli can coexist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.
  • Peltomaa, Elina; Hällfors, Heidi; Taipale, Sami J. (2019)
    Recent studies have clearly shown the importance of omega-3 (-3) and omega-6 (-6) polyunsaturated fatty acids (PUFAs) for human and animal health. The long-chain eicosapentaenoic acid (EPA; 20:5-3) and docosahexaenoic acid (DHA; 22:6-3) are especially recognized for their nutritional value, and ability to alleviate many diseases in humans. So far, fish oil has been the main human source of EPA and DHA, but alternative sources are needed to satisfy the growing need for them. Therefore, we compared a fatty acid profile and content of 10 diatoms and seven dinoflagellates originating from marine, brackish and freshwater habitats. These two phytoplankton groups were chosen since they are excellent producers of EPA and DHA in aquatic food webs. Multivariate analysis revealed that, whereas the phytoplankton group (46%) explained most of the differences in the fatty acid profiles, habitat (31%) together with phytoplankton group (24%) explained differences in the fatty acid contents. In both diatoms and dinoflagellates, the total fatty acid concentrations and the -3 and -6 PUFAs were markedly higher in freshwater than in brackish or marine strains. Our results show that, even though the fatty acid profiles are genetically ordered, the fatty acid contents may vary greatly by habitat and affect the -3 and -6 availability in food webs.
  • de Vera, Caterina R.; Díaz Crespín, Guillermo; Hernández Daranas, Antonio; Montalvão Looga, Sofia; Lillsunde, Katja-Emilia; Tammela, Päivi; Perälä, Merja; Hongisto, Vesa; Virtanen, Johannes; Rischer, Heiko; Muller, Christian D.; Norte, Manuel; Fernández, José J.; Souto, María L. (2018)
    The study of marine natural products for their bioactive potential has gained strength in recent years. Oceans harbor a vast variety of organisms that offer a biological and chemical diversity with metabolic abilities unrivalled in terrestrial systems, which makes them an attractive target for bioprospecting as an almost untapped resource of biotechnological applications. Among them, there is no doubt that microalgae could become genuine cell factories for the biological synthesis of bioactive substances. Thus, in the course of inter-laboratory collaboration sponsored by the European Union (7th FP) into the MAREX Project focused on the discovery of novel bioactive compounds of marine origin for the European industry, a bioprospecting study on 33 microalgae strains was carried out. The strains were cultured at laboratory scale. Two extracts were prepared for each one (biomass and cell free culture medium) and, thus, screened to provide information on the antimicrobial, the anti-proliferative, and the apoptotic potential of the studied extracts. The outcome of this study provides additional scientific data for the selection of Alexandrium tamarensis WE, Gambierdiscus australes, Prorocentrum arenarium, Prorocentrum hoffmannianum, and Prorocentrum reticulatum (Pr-3) for further investigation and offers support for the continued research of new potential drugs for human therapeutics from cultured microalgae.
  • Derman, Yagmur; Selby, Katja; Miethe, Sebastian; Frenzel, Andre; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu (2016)
    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.
  • Brunt, Jason; van Vliet, Arnoud H. M.; Stringer, Sandra C.; Carter, Andrew T.; Lindström, Miia; Peck, Michael W. (2020)
    The neurotoxin formed byClostridium botulinumGroup II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread ofC. botulinumGroup II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverseC. botulinumGroup II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution ofC. botulinumand its neurotoxin genes, dissecting key aspects ofC. botulinumGroup II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.
  • Jokela, Jouni; Heinilä, Lassi M. P.; Shishido, Tania K.; Wahlsten, Matti; Fewer, David P.; Fiore, Marli F.; Wang, Hao; Haapaniemi, Esa; Permi, Perttu; Sivonen, Kaarina (2017)
    Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolandia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A-F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid, which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins, and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 mu M after 1 h) in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic nodularin has the potential to cause the death of eukaryotic organisms.
  • Hietala, Ville; Horsma-Heikkinen, Jenni; Carron, Annelie; Skurnik, Mikael; Kiljunen, Saija (2019)
    The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiMiHyAci03, and Staphylococcus phage vB_SauMiRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_HoEco02 lysate from 3.5 x 10(4) Endotoxin Units (EU)/10(9) plaque forming units (PFU) to 0.09 EU/10 9 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/10(9) PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/10(9) PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.
  • Flores-Rojas, Nelida Cecilia; Esterhuizen-Londt, Maranda; Pflugmacher, Stephan (2019)
    Cylindrospermopsin (CYN)-producing cyanobacterial blooms such as Raphidiopsis, Aphanizomenon, Anabaena, Umezakia, and Lyngbya spp. are occurring more commonly and frequently worldwide. CYN is an environmentally stable extracellular toxin, which inhibits protein synthesis, and, therefore, can potentially affect a wide variety of aquatic biota. Submerged and floating macrophytes, as primary producers in oligotrophic habitats, are at risk of exposure and information on the effects of CYN exposure at environmentally relevant concentrations is limited. In the present study, we investigated CYN uptake in the floating macrophyte Lemna minor with exposure to reported environmental concentrations. The effects were evaluated in terms of bioaccumulation, relative plant growth, and number of fronds per day. Variations in the concentrations and ratios of the chlorophylls as stress markers and carotenoids as markers of oxidative stress defense were measured. With exposure to 25 μg/L, L. minor could remove 43% of CYN within 24 h but CYN was not bioaccumulated. Generally, the pigment concentrations were elevated with exposure to 0.025, 0.25, and 2.5 μg/L CYN after 24 h, but normalized quickly thereafter. Changes in relative plant growth were observed with exposure to 0.25 and 2.5 μg/L CYN. Adverse effects were seen with these environmentally realistic concentrations within 24 h; however, L. minor successfully recovered within the next 48–96 h.